Comparing the catalytic and structural characteristics of a 'short' unspecific peroxygenase (UPO) expressed in P. pastoris and E. coli

DOI: 10.1002/cbic.202200558

Authors: Wendy Robinson (University of York) , Tamara Mielke (University of York) , Benjamin Melling (University of York) , Anibal Cuetos (University of York) , Alison Parkin (University of York) , William Unsworth (University of York) , Jared Cartwright (University of York) , Gideon Grogan (University of York)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Chembiochem

State: Published (Approved)
Published: November 2022
Diamond Proposal Number(s): 9948

Abstract: Unspecific peroxygenases (UPOs) have emerged as valuable tools for the oxygenation of non-activated carbon atoms, as they exhibit high turnovers, good stability and depend only on hydrogen peroxide as the external oxidant for activity. However, the isolation of UPOs from their natural fungal sources remains a barrier to wider application. We have cloned the gene encoding an ‘artificial’ peroxygenase (artUPO), close in sequence to the ‘short’ UPO from Marasmius rotula (MroUPO), and expressed it in both the yeast Pichia pastoris and E. coli to compare the catalytic and structural characteristics of the enzymes produced in each system. Catalytic efficiency for the UPO substrate 5-nitro-1,3-benzodioxole (NBD) was largely the same for both enzymes, and the structures also revealed few differences apart from the expected glycosylation of the yeast enzyme. However, the glycosylated enzyme displayed greater stability, as determined by nano differential scanning fluorimetry (nano-DSF) measurements. Interestingly, while artUPO hydroxylated ethylbenzene derivatives to give the (R)- alcohols, also given by a variant of the ‘long’ UPO from Agrocybe aegerita (AaeUPO), it gave the opposite (S)-series of sulfoxide products from a range of sulfide substrates, broadening the scope for application of the enzymes. The structures of artUPO reveal substantial differences to that of AaeUPO, and provide a platform for investigating the distinctive activity of this and related ’short’ UPOs.

Journal Keywords: Biocatalysis; Oxygenases; Unspecific Peroxygenases; Pichia pastoris; Hydroxylation

Diamond Keywords: Enzymes; Fungi

Subject Areas: Chemistry, Biology and Bio-materials


Instruments: I03-Macromolecular Crystallography , I04-1-Macromolecular Crystallography (fixed wavelength)

Added On: 16/11/2022 11:00

Documents:
ChemBioChem - 2022 - Robinson - Comparing the catalytic and structural characteristics of a short unspecific peroxygenase.pdf

Discipline Tags:

Biochemistry Catalysis Chemistry Structural biology Life Sciences & Biotech

Technical Tags:

Diffraction Macromolecular Crystallography (MX)